The Role of Donor Leukocyte Infusions in the Treatment of Relapsed Acute Leukemia after Allogeneic Stem Cell Transplantation: A Retrospective Analysis.

Background: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only treatment offered for acute leukemias with potential curative capability. One of the main reasons of treatment failure in patients after allo-HSCT is return of the primary disease. This study aimed to evaluate the role of different modalities available to treat the patients with relapsed acute leukemia after allo-HSCT, focusing mainly on donor leukocyte infusions (DLIs). Materials and Methods: This study included 277 patients who relapsed after myeloablative allo-HSCT between February 2003 and February 2015. Treatment option was offered to all patients, but it was not accepted by about one-third of the study participants. Treated patients were categorized based on receipt of DLI (DLI-based vs. non DLI-based). The effect of treatment in all patients and then the effect of DLI among the treated group was evaluated. Kaplan-Meier method was used for calculating survival rates. All patients were relapsed cases, thus only overall survival (OS) was calculated. Results: One hundred and forty-five ALL patients and 132 AML patients were included in the study. One year survival rate for treated patients was 25.13% and for patients who received best supportive care was 2.79% (P<0.001). The difference was significant in both AML and ALL groups. Using DLI-based treatments were accompanied by a noticeably superior outcome. Hazard ratio was 0.43 (0.29-0.63) for DLI-based treatments (P<0.001). Conclusion: Despite the poor prognosis of relapsed acute leukemia after HSCT, it seems that treatment interventions and, especially DLI-based treatments, can be of substantial benefit for patients.


INTRODUCTION
Acute leukemias are among the most frequent hematological malignancies and until now the only treatment offered with a potential curative capability is allogeneic hematopoietic stem cell transplantation (allo-HSCT) 1,2 . Since the advent of more advanced methods to decrease the transplant-related mortality (TRM) mainly from graft-versus-host disease (GVHD) and life threatening infections, one of the deadliest and most difficult outcomes to manage is the relapse of the primary disease 3,4 . Relapse occurs mostly in the International Journal of Hematology Oncology and Stem Cell Research ijhoscr.tums.ac.ir International Journal of Hematology Oncology and Stem Cell Research ijhoscr.tums.ac.ir first 1-2 years after transplant and holds the biggest share of late deaths 5,6 . The prognosis of relapse after allo-HSCT is poor and only a few modalities are available to ameliorate the patients' conditions 7 . These options include aggressive chemotherapy, donor leukocyte infusions (DLIs) and, in some cases, radiotherapy.
Despite promising results in some conditions like chronic myeloid leukemia (CML), DLI was not reported as successful as anticipated in the first experiences for acute leukemia [8][9][10] . The results were particularly disappointing in acute lymphoblastic leukemia (ALL) 11,12 , and until present its efficacy, safety and toxicity is not fully established in acute leukemia. This study was designed with the purpose of evaluating the role of treatment, especially DLI, in relapsed acute myeloid leukemia after allo-HSCT at our center. Patients who were lost to follow-up were contacted to update their survival status. Treatment at our center was planned for all patients, but some of them refused to take it. Therefore, patients were divided into two arms: one arm who received only best supportive care (BSC) and another arm who did receive at least one treatment modality. After survival analysis, the first arm was omitted from analysis and the second arm was classified based on the type and number of treatments. Patients who received DLIs (either alone or in combination with other modalities) were categorized as DLI-based group and non-DLI based group (other treated patients). In order to calculate relapse incidences, the starting point was the time of HSCT and for survival analysis was the time of relapse.

Primary HSCT
The allo-HSCT procedure from full-matched donors at our center was preceded by a myeloablative conditioning regimen. This regimen consisted of busulfan 0.8 mg/kg intravenous (IV) or 4 mg/kg orally (PO) three times a day (TDS) for 4 days (day -6 to -3), and cyclophosphamide 60mg/kg IV daily for 2 days (-2 and -1). GVHD prophylaxis was prearranged by methotrexate 10 mg/m 2 on day +1 and 6 mg/m 2 on days +3, +6 and +11 plus cyclosporine started from day -2 in the following dosages: 1.5 mg/kg/day IV (intravenous) from day -3 to +7, 3mg/kg/day IV from day +8, and then continued until the patient could tolerate oral, then the drug shifted to 6 mg/kg/day PO. If chronic GVHD occurred, the treatment was continued and if no GVHD was present after 3 months treatment was stopped. Folinic acid was administered four times a day (QID) on days 2,4,5,7 and 8 after transplant and two times on day 9. Stem cells were collected from peripheral blood of donors. Body irradiation was not part of the preparation routine.

Diagnosis of relapse and post-relapse intervention protocols
Relapses were screened and diagnosed whenever one of the following events occurred: a decrease in mixed chimerism, minimal residual disease (MRD) detection by molecular or flowcytometry studies, alterations in complete blood count (CBC) or bone marrow aspiration and biopsies in favor of disease recurrence. The first step in the management of for AML patients. When the patients had IEM relapse, radiotherapy (RT) was added to the chemotherapy and if the patient did not accept the chemotherapy, then RT would remain the only therapeutic modality. Treatment efficacy at this step was evaluated by an increase in chimerism, bone marrow aspiration and biopsy (BMA/B). After decreasing the tumor burden and increasing the donor chimerism, patients received DLIs. No appropriate response to primary salvage protocol led to 3 consecutive days of salvage chemotherapy using daunorubicin. DLI process at our center was non GCSF-primed and started from 1x10 7 CD3+ cells. Due to cost considerations, we did not positively select CD3+ cells; therefore, the final infused product consisted of a leukocyte infusion which included the same number of counted CD3+ cells. In responder patients, the same amount was continued for 3 cycles and in non-responders the cell counts were escalated to 2x10 7 cells and then 1x10 8 cells. The time interval between DLIs was one month. GVHD was controlled at the time of DLIs, but in case of active GVHD after DLIs, they were discontinued. By the end of DLIs, mixed chimerism was rechecked by means of PCR. Maintenance of treatment consisted of cytarabine plus interferon-α for AML patients and vincristine plus dexamethasone for ALL patients.

Statistical analysis
The data were analyzed in retrospect and survival rates were computed based on Kaplan-Meier estimate. All studied patients were relapsed cases, therefore only overall survival (OS) rates were calculated. The median follow-up time was calculated using reverse Kaplan-Meier method. The effects of treatment options were analyzed via cox proportional hazard model and compared by logrank test. Variables with a P-value of less than 0.2 entered the multivariate analysis. These variables included treatment, site of relapse for all patients and DLI-based interventions for treated patients. Statistical level of significance was defined as P-values of less than 0.05. Analyses were done in R for windows version 3.2.2 and Stata statistical software (version 11.2).

Basic results and demographics
A total number of 277 patients were included in this study. The mean age at transplantation was 29.83±10.30 years (27.18±9.51 for ALL and 32.74±10.38 for AML). One hundred and ninety patients (68.59%) received treatment and the others received BSC (31.41%). Among the treated group, 57 patients (30%) were treated by DLI-based treatments. The basic characteristics of studied cohort are demonstrated in Table 1.  92%) and a triple combination of DLI, chemo and radiotherapy (11.54%).

Survival analysis
By the end of the follow-up time, 84.12% of patients (n=233) died. Causes of death for all, but two patients, were disease relapse or its complications. Two patients were suffering from severe GVHD after DLIs and eventually succumbed to it. All patients, but 55 with severe GVHD, in the DLI group received 3 DLI cycles with the same dosage explained in the methods. In a median follow-up of 12 months, Kaplan-Meier estimate revealed 1-year overall survival rate of 17.93% for all patients. With regard to treatment, the probability of survival was significantly higher among the treated group than BSC group (   Amongst the patients who received at least one form of treatment, the analysis showed that DLIbased treatments are associated with significant survival advantage compared to non-DLI based treatments (Table2).  Figure 3. Among ALL patients, the same probabilities were 41.47% (95% CI: 18.64% -63.05%) vs. 16.37% (95% CI: 9.04% -25.60%), P=0.03, Figure 4.

Cox proportional hazards model
In the univariate and multivariate analyses of all patients, isolated extramedullary relapse and treatment were significantly related to better consequences. Among the treated patients, DLIbased treatments led to superior outcomes. Results of univariate and multivariate analyses are summarized in Table 3.

DISCUSSION
This study was aimed to evaluate the role of treatment, particularly DLI-based treatments, in the management of relapse in acute leukemia after allogeneic HSCT. As cited earlier herein, first experiences of DLI in acute leukemia were not auspicious starts, nevertheless, more recent prospective studies revealed that DLI-based treatments might potentially have a role in the management of relapse after allo-HSCT (13,14). In spite of a generally poor prognosis of relapsed acute leukemia after allo-HSCT, it seems that treatment interventions could be advantageous for the patients. In our study, the probability of survival for AML patients at first year was 30.79% (with treatment) vs. 2.00% (with BSC). The same difference was also observed among ALL patients (1- 17 . Overall, majority of former studies were not capable of revealing a favorable outcome following intensive chemotherapy in the treatment of relapsed acute leukemia, but the modalities focusing on augmentation of immune responses against malignant cells such as DLI, which supposedly could lead to a stronger graft versus leukemia (GVL) effect and superior outcomes went along with more consideration 15 . A number of existing reports revealed that the decline in donor chimerism of bone marrow B cells, T cells and natural killer cells and also CD34+ peripheral blood cells could herald a hematologic relapse and act as indicator for detection of minimal residual disease (MRD) [18][19][20] .
The retrospective nature of this study, in addition to non-randomized distribution of population in different groups (mainly between treated and nontreated groups), could be a potential hindrance in order to attain a clear and sturdy interpretation of the findings although the multivariate analysis model could adjust and compensate the effect of potential confounders. At our center, we do not perform second transplant which is offered to be efficacious in some studies 21 . The mechanism of action presented for second transplant is to some extent the same as DLI (stimulating the GVL effect), chiefly in cases of changing the donor to an unrelated match. The latter approach has recently came to application 22 .

CONCLUSION
In general, DLI for relapsed acute leukemia is still not working as a potent immunotherapeutic ideal and is not capable of re-induction and maintenance of long-lasting remissions in an acceptable percentage of patients. This could be to some extent due to an imbalance between slow onset of acting for GVL effect of DLIs, the rapid growth of malignant cells in acute leukemias 17 and also the energy of T cells induced by cancer cells that has been shown in some experimental models 23 . Novel techniques such as engineered cytokine-induced killer cells (CIKs) 24 as well as neat strategies to screen the molecular relapse before the presence of an overt hematological relapse are needed to be developed to enhance the efficacy of the procedures and subsequently survival of the patients. Also, developing and usage of modified therapeutic DLIs might lead to better outcomes 25,26 . But, until then, it seems that the necessity of treatment and, especially GVL enhancing modalities like DLI and their survival benefits, should be considered as one of the best existing approaches for an inevitably, extremely fatal disease.